Occasions Glycoproteins for Social and Antisocial A Biochemical Guide to Yeast Adhesins

14 Pages·2007·0.92 MB·English

A Biochemical Guide to Yeast Adhesins:
Glycoproteins for Social and Antisocial
Occasions

Anne M. Dranginis, Jason M. Rauceo, Juan E. Coronado
and Peter N. Lipke
Microbiol. Mol. Biol. Rev. 2007, 71(2):282. DOI:
10.1128/MMBR.00037-06.
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MICROBIOLOGYANDMOLECULARBIOLOGYREVIEWS,June2007,p.282–294 Vol.71,No.2
1092-2172/07/$08.00(cid:1)0 doi:10.1128/MMBR.00037-06
Copyright©2007,AmericanSocietyforMicrobiology.AllRightsReserved.
A Biochemical Guide to Yeast Adhesins: Glycoproteins for
Social and Antisocial Occasions
Anne M. Dranginis,1 Jason M. Rauceo,2† Juan E. Coronado,2 and Peter N. Lipke2,3*
DepartmentofBiologicalSciences,St.John’sUniversity,Queens,NewYork1;DepartmentofBiologyandCenterforGeneStructureand
Function,HunterCollege,CityUniversityofNewYork,NewYork,NewYork2;andDepartmentofBiology,BrooklynCollege,
CityUniversityofNewYork,Brooklyn,NewYork3
D
INTRODUCTION.......................................................................................................................................................282 o
w
StructureofAdhesins.............................................................................................................................................282 n
AWordonAffinities...............................................................................................................................................283 lo
ACTIVITIESOFADHESINS....................................................................................................................................283 a
d
TheAdhesinsofSaccharomycescerevisiaeinMating.........................................................................................283 e
d
S.cerevisiaea-agglutinin....................................................................................................................................283
f
(cid:1)-Agglutinin.........................................................................................................................................................285 ro
Interactionoftheagglutinins............................................................................................................................286 m

Othermatingadhesins.......................................................................................................................................286 h
t
TheS.cerevisiaeFlocculins:AdhesinsforSocialAggregationandForaging.................................................286 tp
TheCandidaalbicansALSFamily.........................................................................................................................287 :/
/
ExpressionprofileandallelicvariationofALSgenes...................................................................................288 m
m
OtherAdhesinsofC.albicans...............................................................................................................................288
b
Candidaglabrata......................................................................................................................................................288 r
.
BROADENINGTHESOCIALLIFE:RECOMBINATIONGENERATESDIVERSITY..................................288 a
s
STRUCTUREOFGPI-ANCHOREDADHESINS.................................................................................................289 m
TheN-TerminalDomains......................................................................................................................................289 .o
CentralThr-RichSequences.................................................................................................................................290 rg
Ser/Thr-RichStalks................................................................................................................................................290 /
o
Pre-GPIandGPISignalSequences.....................................................................................................................290 n

CONCLUSIONS.........................................................................................................................................................290 F
e
ACKNOWLEDGMENTS...........................................................................................................................................291 b
REFERENCES............................................................................................................................................................291 ru
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INTRODUCTION proteinsalsomediatecolonyandbiofilmformation,sotheyare 2
1
“social” from the yeast’s point of view and “antisocial” from ,
Celladhesionproteinsarecriticaltofungalcellinteractions 2
thehumanstandpoint.Suchdualfunctionsarecommon;pro- 0
indevelopment,symbiosis,andpathogenesis.Theadhesinsare 1
teinsthatevolvedtofacilitatecellinteractionsinfungaldevel- 3
locatedonthesurfaceofthecellwall,wheretheymediatethe opmentmayhavebeenexploitedtomediateinteractionswith b
cell’s interaction with the outside world. They participate in y
host organisms in commensal and pathogenic situations (33,
mating, colony morphology changes, biofilm formation, fruit- P
34). Many of these activities have been recently discussed E
ing body development, and interactions with mammalian and (124),sothisreviewwillemphasizethemoleculararchitectures N
plant hosts. These adhesins mediate two types of interaction, N
and binding properties that are used to mediate these func-
which can be thought of as “social” and “antisocial.” The S
tions. T
formertypeisrepresentedbyintraspecificinteractionsinmat- A
inganddifferentiation,suchasthosemediatedbytheSaccha- T
E
romycescerevisiaeagglutininsandCoprinusgalectins(78,128), StructureofAdhesins
U
aswellasformationofcoloniesandbiofilmsthatadheretoand N
invadesubstrata(41,42,71,89,91,110,137,142).Ontheother Yeast adhesins are of necessity mosaic proteins, because IV
theyneedseveraldomainswithdiscretefunctions.Theirlocal-
hand, those interactions we call “antisocial” are typical of
izationontheoutersurfacesofthickcellwallsdeterminesthe
pathogenbindingtohostorganisms.Amongtheseadhesinsin
order of the domains and therefore determines their overall
ascomycetousyeastsaretheEPAgalectinsofCandidaglabrata
architecture. The domains are arranged in a standard order:
andthehydrophobiccellsurfaceproteinsandpeptide-binding
N-terminal secretion signals, domains for ligand binding, op-
AlsproteinsfromCandidaalbicans(15,25,28,33,47).TheAls
tionalcentralThr-richglycosylateddomainsthatfacilitateho-
motypiccell-cellinteractions,N-andO-glycosylatedstalksthat
*Correspondingauthor.Mailingaddress:Dept.ofBiology,Brook- elevate the binding domains above the wall surface, and C-
lynCollege,2900BedfordAvenue,Brooklyn,NY11210.Phone:(718) terminalregionsthatmediatecovalentcross-linkingtothewall
951-5000, ext. 1949. Fax: [phone]. E-mail: plipke@brooklyn
matrix through modified glycosylphosphatidylinositol (GPI)
.cuny.edu.
†Presentaddress:Dept.ofMicrobiology,ColumbiaCollegeofPhy- anchors.Thisreviewwillconcentrateontheproteinsthatcon-
siciansandSurgeons,NewYork,NY10032. formtothismodel(Table1),withparticularemphasisonthose
282
VOL.71,2007 A BIOCHEMICAL GUIDE TO YEAST ADHESINS 283
TABLE 1. Propertiesofyeastadhesins
Adhesin(moleculesper
Species Ligand(affinitya) Knownphysiologicalfunction(s) Reference(s)
haploidcell)
S.cerevisiae a-Agglutinin(104–105)b (cid:4)-Agglutinin(10(cid:3)9M(cid:7)r,p,c(cid:8)) Mating 10,41,52,106
(cid:4)-Agglutinin(104–105)c a-Agglutinin(10(cid:3)9M(cid:7)r,p,c(cid:8)) Mating 10,78,134,141
Flo1p (cid:4)-Mannosides Flocculation 5,112,116,131
Flo5p (cid:4)-Mannosides Flocculation 4,118
Flo9p (cid:4)-Mannosides Flocculation 41,117
Flo10p (cid:4)-Mannosides Flocculation 41,117
Flo11p(106)d Cell-celladhesioninflocs,pseudohyphae, 3,41,72,81,82,101
andbiofilms;agarinvasion;adhesion
toplastic
D
Fig2p Unknown Mating 41,59,138
o
Lg-Flo1p Flocculation 67 w
n
C.albicans Als1p Aggregation,bindingendotheliaand 27,39,47,60 lo
a
epithelia d
Als3p Biofilmformation 90,92,142 e
Als5p (10(cid:3)4Mtosyntheticpeptides(cid:7)c(cid:8)) Aggregation,bindingepithelia 35,49,65,99,107 d
f
OtherAlsproteins 38,39,47,48,50, r
o
51,139,142,143 m
Eap1p Hydrophobicsurfaces Adhesiontoplastic 75,76
h
Hwp1p Substratefortranglutaminase Bindsepithelia 90,110 t
t
p
:
C.glabrata Epaadhesins (cid:5)-Galactosides Bindsepithelia 18,21,61 //
m
aAffinityestimateswerederivedbyradioligandbinding(r),plasmonresonance(p),and/or50%inhibitoryconcentrationforcompetitiveligand(c). m
bMeasuredforS.cerevisiaestrainsW303-1AandX2180-1A. b
cMeasuredforS.cerevisiaestrainsW303-1BandX2180-1B. r.
dStrain(cid:9)1278b(SungsuLeeandA.M.Dranginis,unpublisheddata). as
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for which the most information is available, namely, the Sac- ACTIVITIESOFADHESINS o
n
charomyces cerevisiae agglutinins and flocculins and the Can- F
dida Als proteins. Figure 1A illustrates hydrophobic cluster TheAdhesinsofSaccharomycescerevisiaeinMating e
b
analysis (HCA) of the amino-terminal 440 amino acids of Among the “social” uses of adhesins, those of the benign ru
Flo1p(73).Theaminoacidsequenceishelicallywrapped,and a
breadyeastS.cerevisiaeareprobablythebeststudied.Eachof r
y
residues are color coded by type. HCA analyses highlight re- thetwomatingtypes,aand(cid:4),expressesanagglutininspecial- 2
peated sequences as periodic patterns. Summary diagrams of 1
izedformating.Bindingofthesetwoagglutininstoeachother ,
severalotheradhesinsareshowninFig.1B.Theproteinsare 2
on the surfaces of the mating cells facilitates fusion of the 0
alignedattheirC-terminalGPIanchorsequencestoillustrate haploidcells(78). 1
theirattachmentsitesinthecell. 3
S. cerevisiae a-agglutinin. a-Agglutinin consists of two sub- b
units,theproductsofunlinkedgenesAGA1andAGA2.a-Ag- y
P
glutininhaslittlesimilaritytootherknownadhesins.Aga1pis E
AWordonAffinities
a725-residuepolypeptidewithanN-terminalsecretionsignal N
N
Throughoutthisreviewwedescribebindingaffinitiesasbe- sequence and a C-terminal GPI addition signal (Fig. 1). The
S
ing“low”(equilibriumdissociationconstant[K ]of(cid:2)10(cid:3)6M) remainderisSerandThr-richandhighlyOglycosylated(103). T
(alsocalled“weakbinding”)or“high”(KDof(cid:1)D10(cid:3)6M)(also Unexpectedly, Aga1p is expressed in both mating types, per- AT
called “tight binding”). The weak interactions are character- haps because of its role in agar invasion, as discussed below E

izedbybindingonlywhentheadhesinand/orligandispresent (41). The Aga2p subunit gives a-agglutinin its binding speci- U
N
athighconcentrations,andanyindividualadhesinmaybindto ficity and is expressed only on a cells. Although the mature I
V
its ligand for only a few microseconds to seconds at a time, form has only 69 amino acid residues, Aga2p contains all
because dissociation rates tend to be high. In contrast, high- known binding determinants. This O-mannosylated glycopep-
affinity interactions occur at low adhesin or ligand concentra- tide is doubly disulfide linked to Aga1p (106). The Aga2p
tions,andindividualbindingreactionsmaylastformanymin- half-cystines are near the ends of the peptide, but they are
utes,correspondingtolowerdissociationrates.Therefore,the linked to two Aga1p Cys residues separated by only two resi-
strength and duration of adhesion between yeast cells or be- dues(106).Thus,theclosenessofthedisulfidebondsstabilizes
tween the cell and its substrate vary by many orders of mag- an (cid:4)/(cid:5) structure in Aga2p, similar to small Cys-knot proteins
nitude. In the lab, these differences are often seen as relative (132).
differencesinresistancetovortexingorpipetting.Invivothere Cell surface concentrations of a-agglutinin are low in most
aregreatdifferencesinresistanceofadhesionstoshearforces strains and are increased to about 5 (cid:6) 104 per cell following
suchasflowinthebloodstreamorinbiofilmsinnaturalenvi- treatment with the sex pheromone (cid:4)-factor. The increased
ronments(105,119). surfaceexpressionisaresultofpheromonesignalingthrough
284 DRANGINIS ET AL. MICROBIOL.MOL.BIOL.REV.
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VOL.71,2007 A BIOCHEMICAL GUIDE TO YEAST ADHESINS 285
FIG.1.Molecularfeaturesofrepresentativeyeastadhesins.ThesefeaturesarebasedonHCAplotsoftheadhesins.(A)HCAplotoftheN-terminal440residuesofFlo1p.HCAdrawseachopenreadingframeasahelicalprojection,whichisverticallyrepeated.Individualaminoacidsareplottedandcoloredredforacidic,blueforbasic,andgreenforhydrophobic,withhydrophobicpatchesboundedbyblacklines.Thrresiduesaredrawnashollowsquares,Serasdark-centeredsquares,Glyasdiamonds,andProasredstars.CysresiduesaremarkedwithtrianglesbelowtheHCAplot,andN-glycosylationsitesaremarkedwithmaroonhexagons.TransparentboxesdesignatetheN-terminalsecretionsignal(lightblue)andthebeginningofatandemrepeatregion(openbox).Thelinebelowtheright-handend(cid:2)showsthattheThrcontentoftherepeatregionis25%.(B)SummaryofHCAsofrepresentativeyeastadhesins,alignedattheCtermini,wheretheyarelinkedtocellwallpolysaccharidethroughtheGPIanchorremnant.N-terminalsecretionsignalsarelightblue,andGPIadditionsignalsaregreen.Repeatedsequencesareboxed.Diagonalstripesinwhiteboxesindicatetandemrepeatsthatarenothomologoustootherrepeatsintheillustratedproteins.Wheresimilarsequencesrecur,theyareboxedandtintedinthesamecolor.PotentialN-glycosylationsitesareshownasmaroonhexagonsaboveeachopenreadingframe.Cysresiduesareshownastrianglesbeloweachopenreadingframeandarelinkedwherethedisulfidebondshavebeenmapped.ThecontentofThrisdenotedbybarsbeloweachopenS.cerevisiaereadingframe,whicharedottedwheretheThrcontentexceeds20%andsolidwhereitexceeds25%.(Thefrequencyintheentiregenomeis6%.) taomsc(cp(cid:4)d(iaatbwmpi1t1m1idcfahemb4(cid:10)nnnoeeie11ree00-lrnfl0-ynruii-oheegtfi(cid:11)4werauetcgaaane204F0ollaC(cid:1),eddtcemnmnlldtpiwnhtam-gtmn9,hdygmIe(cid:6)raeci7fairt-i-,Gnseegi(lvna)eoooglscsnAhiadstt71iyrotselFa.anptleneanlhlusi1ng-.whycgu,0gdtlwuselelctTaitr0engiociltrimteyaia6ggtn2icsfot1mhs1lic(cid:3)lftirosltgdidhriiocf.t..l)htaysutn3eyb0aofignri4yuagfeae.hreepcicncocel2p4a(cid:3)AiruanrtuincldlaetMasn1oxernlhatsnaose;,Tsmenilcrbsalse3iddho0rpot,snieeec,tbtoo,.1dTelnhbop0hgoayieuymls,ratMdlilutuTrsf4a,wiseteeel(cid:4)ofilnteorapbpi0kayss(tlho0enrsoshCrtttxsb.w0up.ihaeIo-epcettihhhiio)iwtdsncynlg0erag(cid:4)tlrrzro-seprliloeea-.ineeeeloat-nyoea)cednnahrtp-nctmsDledrSd-hbt,n“hlsAliegdwfou(cid:4)iwegke1lre,ardfimufiiuueeitasinrboairnmicnt-)igckdhgaoemcnnflxcretntoaaclooemettataidehgeenrhsofstanieh(eiotgfguldrcdciaiet.dlntifilh1denncriitpiuigyuenfaen4hpoToccn.nneomaat0steontwtsrlt0utkeger.hcsfchidloouilulgia6Tilioaanyapsnnspooicncdeettmeaet)iNrthlpAerftoe-eoyntnealnitalii.slcdrnoebclienesonne”ghcsl,ngseicoe,u(cid:4)gecisaS-sClurg/niedpsueigvasstoasmldalinlltca-ndunaitlisterudeatt-elrhnanicsi2noscruiaiutndtnldsscwnrfrtubhrtdcgbeardgpaetpniefalhhirtigdotaeoinievakgaarauedvrnxilhfhsotnctaftnaitnewcmwsmaloalarh.aitivihseihenudtylmets(ocbeeeigcgntosrlrheTaeilsint1eistshienaageei(agench.snontaidotn0taocehdc6arttoenAhsowimvoiaipgiohlice,odrfdlscrshsa4neonoieooflalposiltn,foiid1oscfnc)pinlatlpbaoiltsnnfigcrnlarefamf.ncvoea3espssdomorf,iooaugec,ahrnnuecediunco,cdpnTecteelinigat,ctrsstuer-delnietbhndrhc7klpelrmatfi(olltiehl4rnolsanaooaouesneeeity8dCeirvifbgienxfwnhtttnficte(cid:11)nbnhrvbngro,entiairiniaoDretgasroomarnemditsuamaitc1oAcwakdinew,fprtrlcnhcnlivzte)ltl3(tahipiaiehet,tdoitsarii,egenei(cid:10)e(cid:4)ngtsitytao4onoertagaoeonrsiehihaodsoaaaotinsf-)tgnntmn1inbncpoCleifi2aaelhnso.vthcyAagd0nengtiaessrenesbeincpgtgendihebW0nrhcsepgelewsragvotsuygda,eayoarsteopvqice,cnreasorpnbrnlslna1d1uciewtewuweaarauu(cid:4)sar2oeAom-cioptte0msdmtiiahtattyns(cid:11)nsailprtbsvbmleeixafhn62opsylgit)iuocadcnmiplcuisctn.s(wwpflasolnnl5opidehdatttiieideof1roaernemioiot2hhenmiauoldeerpbeteenyrntoc3(cid:6)rwflsiidnpiaeeeghye.yrssrss-------t-----tf,, on February 21, 2013 by PENN STATE UNIVhttp://mmbr.asm.org/Downloaded from
have been identified and mapped on the model (19). The
Ig-like region of (cid:4)-agglutinin is conformationally flexible: the
nativestatehasahighpercentageof(cid:5)-sheet(45%)andabout
2% (cid:4)-helix (13). The (cid:5)-sheet content increases upon ligand
286 DRANGINIS ET AL. MICROBIOL.MOL.BIOL.REV.
binding,andthe(cid:4)-helixcontentincreasesunderenvironmen- Othermatingadhesins.Thea-agglutininGPI-anchoredsub-
tal stress (140). Interestingly, the globular regions of the ho- unit Aga1p has another adhesive role as well. While Aga1p
mologousAlsproteinshavebeenmodeledmorerecently,and functions as the anchorage subunit of a-agglutinin, it is ex-
Ig-liketemplateswerealsousedinthatstudy(107). pressed in both mating types. Aga1p or the similar protein
Interaction of the agglutinins. Because both (cid:4)-agglutinin Fig2p is essential for completion of mating and for mating-
anditsliganda-agglutininhavebeenextensivelycharacterized, dependentinvasionofagar(41).Littleisknownaboutwhether
wecandescribeamodelofthebinding.Bindingof(cid:4)-aggluti- theseproteinsactdirectlyasadhesins,butFig2phasarolein
nin to high-affinity peptide epitopes on the a-agglutinin bind- signaltransductiontothewallstresspathwayandthereforehas
ingsubunitAga2pisslowandtight(K of(cid:10)10(cid:3)9M)(10,106, aroleinmaintenanceofcellwallintegrityduringmating(59,
D
141).Theagglutininsaredisplayedontheoutersurfaceofthe 138). It is pheromone induced and has an indirect role in
cellwall(10,133)at104to5(cid:6)104copiespercell,correspond- attenuating sexual agglutination in mating type (cid:4) cells (59,
D
ing to concentrations of 10 to 100 (cid:11)M in the 100-nm space 138).Thus,Aga1pandFig2parepartiallyredundant,andeach o
betweenthecells(Fig.2)(106).Atsuchconcentrations,both has multiple adhesive roles. Such manifold activities and par- w
n
high-affinitybindingandlow-affinitybindingarerapid.There- tialredundancyarecommonamongtheyeastadhesinsingen- lo
fore, cell surface adhesion assays show interactions with mi- eral. a
d
cromolarorevenlargerdissociationconstants.Incontrast,in e
d
vitro binding assays such as surface plasmon resonance or
TheS.cerevisiaeFlocculins:AdhesinsforSocial fr
125I-protein binding assays can document only much tighter o
AggregationandForaging m
interactions, with affinities in the nanomolar to micromolar

h
range. Indeed, the agglutinins bind with both nanomolar and Many adhesins mediate interactions between yeast cells to t
t
p
micromolar affinities (106, 141). Because the C-terminal resi- form aggregates (flocs). These interactions have been called :
/
dues of Aga2p are the major binding determinant, a surface “flocculation”or“aggregation.”Wewillusetheformertermto /m
display model that fuses peptides to be screened to the C describe Ca2(cid:1)-dependent lectin-mediated interactions. Floc- m
terminus of Aga2p inactivates the high-affinity binding (106, culation and aggregation are asexual; that is, they can occur b
r
.
129).Therefore,itisthelow-affinityinteractionsthatareused amongcellsofanymatingtypeandarenotpartofthemating a
s
inscreening.Thisresultandmutationalanalysesdemonstrate response.Theseinteractionsareintegraltobiofilmformation m
thatthebindingiscomplex,involvingseveralregionsofAga2p (101). .o
withseveralregionsofAg(cid:4)1p(106). S. cerevisiae harbors a family of flocculin genes with five rg
/
In addition, conformational shifts in the agglutinins are es- members:FLO1,FLO5,FLO9,andFLO10(117)andFLO11 o
n
sential for effective binding, even in this well-characterized, (72,82)Ofthese,onlyFLO11isexpressedinmostlaboratory
F
apparently simple reaction (141). Binding of the S. cerevisiae strainsofS.cerevisiae,whereitexhibitsaprofusionofpheno- e
b
agglutininsiskineticallyirreversible,duetoverylowdissocia- types.FLO11expressionisrequiredforflocculationinS.cer- r
u
tion rates (hours to days) (80, 141). This irreversibility would evisiae var. diastaticus (82) and for invasion of substrates and a
facilitate stable binding of partnered cells in mating and pre- formation of pseudohyphae in (cid:9)1278b strains (72, 81). Floc- ry

sumablyresultsfrominteractionsofindividualmoleculesthat culationisimportanttothebrewingindustry:Yeastcellsfloc- 2
1
includefairlylargesurfaceregionsofeachofthetwoaggluti- culate spontaneously at the end of fermentation, with the re- ,
2
nins(19). sult that the majority of cells are separated from the culture 0
1
Such kinetic irreversibility is also characteristic of systems medium(122). 3

withmultipointattachmentsatloweraffinity,suchasfloccula- TheFlo11pflocculinalsoenablesyeasttoadapttoachang- b
y
tionorbindingofAlsproteinstomanyligandsonamamma- ing nutritional environment by switching to a pseudohyphal P
liancellsurface.Inthesecases,multipointattachmentsformas modeofgrowth.Thefilamentouschainsofpseudohyphae(Fig. E
N
aresultoftheextremelyhighcellsurfaceconcentrationsofthe 3C), which may invade the substrate, develop in response to
N
adhesins,manyofwhichareboundtoligandsthatarealsoat starvationfornitrogenindiploidcells.Thismodeofgrowthis
S
high surface concentrations. For instance, flocculins bind to consideredanadaptationforthisnonmotilespeciestoforage T
A
mannose residues, and Als proteins bind to common peptide for nutrients (36, 37). Pseudohyphal differentiation involves T
sequencemotifs,bothwithmillimolaraffinities.Mannoseres- changesingenetranscriptionandcellcycleprogressionaswell E

idues can be at molar or higher concentrations on the cell as changes in cell morphology (reviewed in reference 30). U
N
surface. If a cell is attached at two points on its surface, the Flo11pmayplayaroleinpolarizingcellshapeinadditiontoits I
V
dissociationrateofthecellsdecreasesandtheapparentaffinity roleinadhesion(93).TheFlo11padhesinisrequiredtohold
increases as the product of the individual values. Therefore, the cells together in the branched chains. Thus, adhesin ex-
millimolaraffinitiesbecomemicromolarwithtwoattachments, pressioncangoverncolonymorphologyandinvasion.
andnanomolarwiththreeattachments,betweentheyeastand Flo11palsoenableshaploidyeasttoinvadeagarinresponse
themultivalentligandorsubstrate.Ifthedissociationratefor toglucosestarvation(17)oraminoacidstarvation(8).Adhe-
thebindingwas10(cid:3)1s(cid:3)1,thecorrespondingwhole-celldisso- sion to the substrate is the essential step in invasion (41).
ciationtimeswouldbeabout10sforasingle-pointattachment, Presumably,invasionistheneffectedbytheforceofcelldivi-
1.5 min for two attachments, and 15 min for three-point at- sion from the adherent cells. In addition, FLO11 is required
tachment. In this way, a high cell surface concentration of fortheformationofbiofilmstructuresonagar(Fig.3B)(101),
low-affinityadhesinsandalowerconcentrationofhigh-affinity foradhesiontoplastic(101),andforformationofthespecial-
adhesins yield similar energetics and longevity of cellular ad- ized floating biofilms called flors that are responsible for the
hesions. production of sherry wine (24, 54, 55, 137). The diverse phe-
VOL.71,2007 A BIOCHEMICAL GUIDE TO YEAST ADHESINS 287
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FIG. 3. Several phenotypes associated with the adhesin Flo11p. (A) Flocculation of S. cerevisiae var. diastaticus is Flo11 dependent. Yeast /m
culturesofequalcelldensitieswerevortexedvigorouslyandphotographedattheindicatedtimeintervalsaftermixing.Lefttubes,wild-typehaploid m
cells;righttubes,isogenicyeastwithFLO11deletion.(B)S.cerevisiaestrain(cid:9)1278bformFlo11-dependentbiofilms.Asinglecolonyofyeastis b
showngrowingonsemisolidmediuminastandard100-mmpetridish(101).ThebiofilmwithitscharacteristicfloralstructurerequiresFlo11for r.
itsformation.(C)Diploid(cid:9)1278bstrainsformbranchedchainsofcellscalledpseudohyphaeinresponsetonitrogenstarvation.Formationofthese as
chainsrequiresFlo11p(81). m
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g
/
notypes of FLO11 could be explained as a consequence of invasion,likethatcausedbyFlo11p.OverexpressionofFlo1p o
n
altered adhesion in diverse circumstances. How different does not cause agar invasion but does promote flocculation.
F
strains of yeast can exhibit different FLO11-dependent prop- Overexpression of Flo10p can promote both flocculation and e
b
ertiesisamysterystillunderinvestigation.Forexample,theS. Flo11p-like pseudohyphal development (41). Therefore, the r
u
cerevisiaevar.diastaticusstrainrequiresFLO11forflocculation rolesoftheflocculinsareoverlappingbutnotidentical,andthe a
(82)(Fig.3A),while(cid:9)1278bstrainsthatexpressFLO11donot flocculinfamilymembershavepartialfunctionalredundancy. ry

flocculate(41).Clearly,otherfactorsmustbeinvolvedinthese The flocculins are lectins, proteins that bind to cell surface 2
1
phenomena.Potentialcausesmightbesequencedifferencesor carbohydrates. Each yeast lectin is specific for one or two ,
2
differentialglycosylation. monosaccharide haptens (e.g., for mannose or for mannose 0
1
Flo1pandFlo5p,whichare96%similar(117),wereidenti- and glucose) but binds to haptenic oligosaccharides that con- 3

fied as proteins that cause flocculation (4, 58, 116, 130, 131). tain these sugars in several different glycosidic linkages. Con- b
y
They are large proteins that display structures typical of floc- sequently,oligosaccharideswithspecificsugarindiverselink- P
culins: a hydrophobic N terminus with signal sequence, a hy- ages can act as hapten inhibitors (61, 124). Low specificity is E
N
drophobicCterminuswithaGPIanchorconsensussequence, usually accompanied by low affinity (millimolar or higher for
N
andacentraldomaincomprisingtandemrepeatsofsequences monosaccharides and disaccharides) (56), because binding
S
thatareextremelyrichinThrresidues(Fig.1).Thesearecell sites are broad and flexible, whereas high-affinity sites sur- T
A
wall lectin-like proteins that participate directly in adhesive round one specific kind of structure and maximize the inter- T
cell-cell interactions (4–6, 67, 118). Flo1p and Flo5p, along actionswithallsurfacesoftheligand.Broadspecificitywould E

with Flo11p, have been identified as “Flo1-type flocculins” allow binding to many structures on the surfaces of host cells U
N
whose activity is inhibited by mannose but not by glucose (3, andonotherfungi. I
V
108,112).Avariantflocculin,Lg-Flo1hasbeenidentifiedasa
memberofthe“NewFlo-typeflocculins,”aclasswhoseactivity
TheCandidaalbicansALSFamily
is inhibited by both mannose and glucose. Domain swap ex-
perimentshaveestablishedthatthesugar-bindingdomainsof The C. albicans ALS adhesins have both “social” (aggrega-
Flo1p and Lg-Flo1 are the externally exposed N-terminal do- tive) and “antisocial” (pathogenic) functions. They have N-
mains(67). terminalIg-likedomainshomologoustotheS.cerevisiaesexual
FLO9 and FLO10 were identified on the basis of their se- adhesin(cid:4)-agglutinin(compareAlsproteinsandSag1pinFig.
quencesimilaritytoFLO1andwerefoundtoalsocausefloc- 1B)(47,107).Alsadhesinsmediateadhesiontoepithelia,yeast
culationwhenexpressed(41,117).TheFLOflocculinsdifferin aggregation, and biofilm formation. The family is encoded at
their functional roles, but when overexpressed some can sub- eight loci, and each locus is heterozygous (144). The various
stitute for others. Expression from a galactose-inducible pro- formsareexpressedatdifferentphasesofgrowthandinfection
moter of either Fig2p or Flo10p in strain (cid:9)1278b causes agar (47, 143). Because of the widespread expression of different
288 DRANGINIS ET AL. MICROBIOL.MOL.BIOL.REV.
AlsproteinsinC.albicans,functioncanrarelybeassignedto OtherAdhesinsofC.albicans
asinglememberofthefamily.Therefore,mechanisticstudies
Severalhydrophobiccellsurfaceproteinsincreasepartition-
have relied on heterologous expression in S. cerevisiae . In all
ing of yeast cells into organic solvents in water/solvent inter-
known cases, Als-dependent behavior in S. cerevisiae mimics
faces.Ofthese,C.albicansEap1pmediatesbindingtoplastic
thatinC.albicans,andALS1andALS5wereclonedbasedon
surfaces in a manner resistant to shear forces (75). These
their ability to mediate S. cerevisiae adhesion to mammalian
propertiesarethoserequiredforstableadhesiontoindwelling
cells(27,33,107).Als1pisthemostwidelyexpressedmember
catheters and other devices, so the protein may be critical in
of the family and contributes to adhesion and colonization in
prosthesis-induced candidemias and endocarditis. Eap1p has
an oropharyngeal candidiasis model (60). Als1p binds to en-
the same general architecture as the GPI-cross-linked lectins
dothelia and epithelia. Als3p is important for biofilm forma-
and peptide-binding proteins (Fig. 1). A hydrophobic-sub-
tionandinductionofdamageinanunderlyingoralepithelium strate-bindingproteincalledCsh1phasbeenextensivelyfunc- D
(71, 90, 91, 100, 107, 142, 143). For the Als proteins, the tionally characterized in S. cerevisiae and, surprisingly, shows ow
N-terminal globular domain is necessary and sufficient for sequencesimilaritytoknownglycosyltransferases(45).Csh1p n
bindingtocellsurfaces(85,99,107).Thebindingspecificityis fromCandidadublinensisfunctionssimilarlyandhasasimilar loa
extremely broad, and both Als1p and Als5p bind to peptides distribution of hydrophobic residues in HCA analyses (not d
e
containing a common structural motif: (cid:12)(cid:13)(cid:1) (a turn-like resi- shown).Nevertheless,BLASTsearchesshowitsgenebelong- d

due,abulkyhydrophobicresidue,andLysorArg).Thismotif ing to a different gene family; it is homologous to aryl oxi- fr
o
ispresentinmultiplecopiesinmostproteins.Als5pandAls1p doreductases and K(cid:1) channel regulatory domains (46). Nei- m

show overlapping specificities but show some differences in ther of these Csh1p proteins possesses a secretion signal or a h
t
bindingtoindividualinstancesofthemotif(65).Peptideswith GPIadditionsignal.Rather,theyarerepresentativeofanum- tp
:
other sequences also bind to Als proteins, making binding ber of cytoplasmic metabolic enzymes that have historically //
m
specificity even broader (32, 35). Flexibility of the peptide beenreportedascellwallconstituentsaswell.Littleisknown m
backboneisessentialforbinding,whichisdisruptedbyH-bond of their localization or biological roles (45, 46, 66, 109). A b
r
perturbants (35). The apparent affinity to such peptides is numberofhomologsofeachoftheseproteinsappearintheC. .a
around 0.5 mM. Thus, Als1p or Als5p causes expressing C. albicansgenome. sm
albicanstoadheretoendotheliaandepithelia,aswellasbio- C. albicans Hwp1p, a glutamine-rich, GPI wall-anchored .o
chemicallydefinedsubstrates. adhesin, is a substrate for epithelial cell transglutaminases rg
Expression profile and allelic variation of ALS genes. The (110, 111). Thus, it participates in a covalent cross-link be- o/
tween the yeast and the epithelium. The resulting association n
ALS genes are differentially expressed in different growth
would be shear resistant, extremely close, and permanent in F
phasesandindifferentmorphologicalforms(38,48,50,51). e
theabsenceofproteolysis.Suchinteractionscouldunderliethe b
ALS1expressionismaximaljustafterinoculationintofresh r
clinicalobservationthatoropharyngealcoloniesofC.albicans u
growth medium (143). Similarly, ALS3 expression is maxi- a
resistremovalbyscraping(89). r
malwhengermtubesaremicroscopicallyvisible,anditmay y

2
mediatethewell-knownautoaggregationofgermtubes(90, 1
,
143). Candidaglabrata 2
ALS gene activity accompanies C. albicans pathogenesis. 0
TheC.glabrataEPAlectinsaremembersofthesamegene 1
ReversetranscriptionPCRtestsdetectedALSgeneexpression 3
familyastheflocculins(BLASTEvalueof10(cid:3)11)andarealso b
inhumanclinicalspecimensandinavaginalcandidiasismodel Ca2(cid:1)-dependent, but they bind (cid:5)-galactosyl- and (cid:5)-N-acetyl- y
(14).AlthoughtranscriptionfromallALSgeneswasobserved, P
galactosaminyl-containing saccharides (15, 18). Therefore, E
ALS1,ALS3,andALS9weredetectedmostfrequently.Similar they are members of the galectin class of sugar-binding pro- N
resultsforALStranscriptionalactivitywerefoundinamurine N
teins. The EPA gene family encodes adhesins which mediate
model of disseminated candidiasis (38). Experiments utilizing S
bindingtohostepitheliaduringinfections.Whileexpressionof T
the gene encoding the yeast enhanced green fluorescent pro- EPA1,EPA6,andEPA7isrepressedinthecirculationofmice, A
tein as a reporter gene under control of ALS promoters sug- T
these genes are specifically induced in the urinary tract, en- E
gested that some Als proteins (mainly Als1p and Als3p) are ablingC.glabratatocolonizethebladder(21).Themechanism U
abundant on the C. albicans cell surface, while others are of gene expression involves relief of telomeric silencing. The N
I
producedatlowerlevelsinthemousedisseminatedcandidiasis low nicotinic acid concentrations in urine lead to reduced ac- V
model(39). tivity of the NAD(cid:1)-dependent histone deacetylase Sir2p,
Like the flocculins, Als proteins can aggregate cells that which is necessary for maintenance of silencing. Levels of
expressthem.Als-mediatedaggregationdoesnotrequireCa2(cid:1) EPA4andEPA5werealsoobservedtorisemodestlyinurine.
andsoisdifferentfromflocculation,whichisCa2(cid:1)dependent. In vitro, EPA6 has been shown to be involved in biofilm for-
However,theThr-richdomainsofAls5pareclearlyinvolvedin mation(53).
cell aggregation in C. albicans (99). Therefore, the Als ad-
hesins mediate both adhesion to mammalian tissues and ag-
BROADENINGTHESOCIALLIFE:RECOMBINATION
gregationoftheC.albicanscellstoformmicroscopiccolonies.
GENERATESDIVERSITY
In parallel with a recent finding for S. cerevisiae FLO1 (123),
AlsproteinswithmorecopiesoftheThr-richrepeatsaremore Many of the adhesins include central-region direct repeats
aggregative(47,99). thatareThrrich(Fig.1).Suchrepeatsarehighlyvulnerableto
VOL.71,2007 A BIOCHEMICAL GUIDE TO YEAST ADHESINS 289
recombination, enabling the reshuffling of protein domains. This switch is regulated by the histone deacetylase Hda1p
The shuffling has led to diversity in adhesin structures and and is heritable for several generations. The silent FLO10
activities,aswellasto“invention”ofnewproteins(123).InS. geneinthesecellsisalsoheterogeneouslyexpressed,dueto
cerevisiae, four of the five FLO genes and their pseudogenes high-frequency mutations in IRA1 and IRA2. Silencing of
areadjacenttotelomeres,achromosomallocationthatispar- FLO10isaccomplishedbyadifferentsetofhistonedeacety-
ticularly susceptible to recombination. One such recombina- lases, Hst1p and Hst2p (43). The consequent cell surface
tion event between the repeated sequences of FLO11 and heterogeneity of yeast populations means that they are
SGA1 appears to have given rise to the STA gene family of primed for rapid adaptation to changes in environmental
secreted glucoamylases (72, 82, 126, 135). The STA family conditions,becausetherearepreexistingadhesiveandnon-
comprises STA1, STA2, and STA3, all of which are telomere- adhesive subpopulations.
associated glucoamylase genes which are present only in the
Thus,therepeatsequencesgenerategeneticdiversityinthe D
variant strain S. cerevisiae var. diastaticus (98). This variant flocculins through recombination, while epigenetic mecha- o
yeast strain is defined by the presence of one of these genes. w
nisms provide the capacity for rapid adaptation to changing n
The genetic recombination that presumably gave rise to the conditions.Giventheshortgenerationtimeoftheseunicellu- lo
STAgenesresultedinthefusionoftheamino-terminus-coding a
larorganisms,apopulationofyeastisthuscapableofrapidly d
sequences(includingthesignalsequenceandpartoftheThr- e
changingitsadhesivepropertiestopermitcolonizationofnew d
rich repeats) of FLO11 with the glucoamylase-coding se-
niches.Adaptiveradiationofthissortmayexplaintheperiodic fr
quencesofSGA1.Theresultingproteinisaglucoamylasewith o
outbreaksofpathogenicstrainsofS.cerevisiaeinpatientswhen m
a FLO11 signal sequence and is secreted. S. cerevisiae var.
thisusuallyharmlessbreadyeastisusedasaprobiotictherapy h
diastaticus has been isolated on several occasions in different t
tocontrolantibiotic-induceddiarrhea(11). tp
partsoftheworldfromoverfermentedbeer(1,62,113,114). :
/
The ability to secrete glucoamylase enables the variant strain /m
to utilize starch after other carbon sources have been ex- m
hausted. Thus, recombination of the Thr-rich repeats enables STRUCTUREOFGPI-ANCHOREDADHESINS b
r
.
secretion,whichconfersaselectiveadvantageoveryeastwith- a
The GPI-anchored adhesins share a common architectural s
outtheSTAgene. m
plan (Fig. 1), so we will discuss the various regions starting
SeveralrepeatedmotifsintheFLOgenesareconservedin .o
fromtheNterminus.Inorder,theyareasfollows:asecretion r
the DNA sequence as well as in the amino acid-coding se- g
signal, a globular adhesion domain, an optional Thr-rich re- /
quence, providing further evidence that homologous ex- o
peat domain, a glycosylated Ser/Thr-rich “stalk,” the prean- n
changesatthesesequencesmayprovideaselectiveadvantage
chorregion,andtheGPIadditionsignal.ThelengthsofGPI- F
to the organism (125). Recombination between FLO5 and a anchored adhesins range from 650 residues ((cid:4)-agglutinin) to eb
flocculinpseudogenemayhavegivenrisetotheNewFlo-type r
about1,650residuesforsomeoftheAlsproteins,Flo11p,and u
flocculin Lg-Flo1p (67). The fact that most of the genes in a
Fig2p. r
the S. cerevisiae genome that contain repeated sequences y

encode cell wall proteins illustrates the functional signifi- 2
1
cance of these domains (123). ,
TheN-TerminalDomains 2
The ALS adhesin genes of C. albicans also contain central 0
1
domains of 108-bp tandemly repeated Thr-rich sequences, 3
N-terminalsecretionsignalscomprise20to30aminoacids
which exhibit considerable allelic variation. The number of b
andarecleavedbysignalprotease(10,13). y
these repeats varies considerably between alleles of a given Theglobularadhesiondomainsfollowthesignalsequences P
ALS gene, resulting in a large repertoire of adhesins (47, 92, E
andcontaintheknownligandbindingsites.InS.cerevisiaethe N
144).Remarkably,60differentallelesofALS7werediscovered
(cid:4)-agglutinin N-terminal region has the sedimentation coeffi- N
in an analysis of 66 clinical isolates of C. albicans. The allelic
cientofaglobulardomainandshowsaCDspectrumtypicalof S
differenceswereprimarilyduetorearrangementsinrepeated T
(cid:5)-sheet-rich folds (115, 140). Residues important for binding A
motifs(139).ThegeneticandallelicvariabilitiesofALSgenes T
to Aga2p are in this region and are localized in loops that
leadtodifferencesincellularbehavior.Studiesexaminingthe E
two ALS3 alleles showed a drastic difference in adhesion to connect (cid:5)-strands in the Ig-like structural model (19). In C. U
albicans the Als protein N-terminal domains are homologous N
vascular endothelial cells; the larger allele (containing a to (cid:4)-agglutinin and so also have Ig-like structures. Als1p and IV
greaternumberoftandemrepeats)conferredmuchmoread-
Als5palsohave(cid:5)-sheet-richCDspectra,andpredictionshave
herence than the smaller allele (92). Further analysis of ALS
allelic pairing across five major C. albicans clades demon- beenmadeforligand-bindingregions(49,107).
strated a tendency of C. albicans to encode one smaller and Other adhesion domains have not been physically studied,
onelargerALSallele(92). but secondary-structure studies are intriguing. CD spectros-
Epigenetic regulation of flocculin gene expression provides copyshowsthatAga2phasboth(cid:5)-sheetand(cid:4)-helicalregions
additionalvariationincellsurfaceproperties.WhendiploidS. (106).TheFlolectinsarealsopredictedtobe(cid:5)-sheetrichby
cerevisiae cells are starved for nitrogen they develop as secondary-structure predictors. The prevalence of (cid:5)-sheets in
pseudohyphae, but this response is not homogenous. Some thissmallsampleofadhesinsmaybefortuitous,butitmightbe
cells remain in the yeast form, which is the single-cell form. a consequence of the acid stability of (cid:5)-sheet structures in
These yeast form cells have undergone an epigenetic switch environmentstypicalforgrowthoftheseacidogenicorganisms
thatresultsinthemetastablesilencingoftheFLO11gene(43). (140).
290 DRANGINIS ET AL. MICROBIOL.MOL.BIOL.REV.
CentralThr-RichSequences Pre-GPIandGPISignalSequences
Many of the adhesins, including Flo lectins, Als adhesins, GPI-linkedproteinsinfungiareintwoclasses.Some,typi-
andEap1p,havetandemlyrepeatedcentralregionsofseveral fied by Gas1p, are primarily membrane proteins and tend to
hundred residues that are rich in Thr and other (cid:5)-branched remain with their GPI fatty acids embedded within the mem-
amino acids (Fig. 1). Such amino acids have a strong prefer- branebilayer.Ontheotherhand,theadhesinsandotherwall
ence for extended, (cid:5)-sheet-like structures. However, the Pro proteins,suchasCwpproteinsinS.cerevisiae,losetheirasso-
residuesalsocommoninthisregionmaypreventformationof ciation with the membrane, and become covalently cross-
compactdomains,aswouldthemanyOmannosylations,which linkedtothewallglucans.In(cid:4)-agglutininandotherGPIwall
arepresentintheThr-richregionsofAls5pandFlo11p.These proteins,atruncatedGPIglycanbecomestransglycosylatedto
regionshavebeenproposedtofunctionasspacersinAlspro- (cid:5)-1,6-glucan(63,69,79).
teinsandinflocculins,holdingtheadhesiondomainawayfrom There is significant research addressing the question of D
o
the wall (85). However, they also play additional roles in ad- whetherthereisalsoasignalthatspecifiesthataGPImoiety w
hesion(99). will be directed to the wall or remain membrane attached. nlo
The Thr-rich repeats account for about 70% of the amino Investigationofthisquestioniscomplicatedbytheobservation a
d
acidsofFlo1.Theserepeatsplayaroleindeterminingfloccu- that GPI proteins are always found to some extent in both e
lation levels. In the flocculins and Als adhesins, proteins with locations (16, 87, 88). Indeed, recent information shows that d
f
fewerrepeatsdisplayreducedflocculationlevels(5).Whenthe GPI-linkedwallproteinsareoftencross-linkedintothewallby ro
numbersoftandemrepeatsinFLO1wereexperimentallyma- other mechanisms as well, including glutamine-dependent m

nipulated, a linear relationship was found between repeat transesterification(22,64)anddisulfidebondformation(52). ht
number and flocculation as well as strength of adhesion to The result of these complications is that the proportion of tp
:
polystyrene (123). Similarly, the aggregation ability of the C. proteins localized within the wall has usually been underesti- //m
albicansAlsproteinsisroughlyproportionaltothenumberof mated, a result of incomplete release of the wall-bound pro- m
Thr-richrepeats(99),andtheAls5prepeatscanmediatecel- teinsaswellasbecauseofpoorelectrophoresisandblottingof b
r
lularaggregation(99).Therefore,itislikelythatmanyofthese yeastproteoglycans(88).Nevertheless,thesequenceimmedi- .a
ately before the GPI addition signal influences the ratio of s
repeatshaveadhesiveactivityaswellasbeingrecombinogenic. m
membrane-associated to wall-associated protein (9, 44). Spe- .
o
cifically,hydrophobicresiduesatpositions4and5beforethe r
g
Ser/Thr-RichStalks GPI transpeptidylation site direct a greater proportion of the o/
protein to the wall (9), and basic residues at one and two n

TheSer/Thr-richregionsareatleast300residueslong,with residuesbeforethissitedirectmoreproteintothemembrane F
e
35 to 55% Ser and Thr residues. In the few cases where they (64). HCA plots show that all “wall-directed” proteins have b
r
have been analyzed for carbohydrate content (the Aga1p an- substantial hydrophobic regions four to eight residues N ter- u
a
alogfromHansenulawingei,thefirst25residuesoftheSer/Thr minal to the GPI addition site, whereas the “membrane-di- ry
regionin(cid:4)-agglutinin,andasimilarregionneartheCtermi- rected” proteins show an exclusion of hydrophobic residues 2
1
nus of the GPI membrane protein Gas1p), all Ser and Thr withinabout20residuesofthesite(notshown). ,

2
residues have been found to be O glycosylated (13, 31, 136). 0
Thus,theywouldbepredictedtoformanextendedconforma- 1
CONCLUSIONS 3
tion (57). Such an extended structure is consistent with the b
y
predictedlengthofthestalkvisibleinanelectronmicrograph Yeastsarenonmotileeukaryotesthatrelyontheiradhesins P
of wall-bound (cid:4)-agglutinin (10, 78). This length would allow for selective interaction with the environment and with other E
the ligand-binding regions of Epa1p, Als1p, and Aga2p to be cells.Wehavereviewedthestructureandbindingpropertiesof N
N
displayedfarenoughfromthewallsurfacetobeactive(78).In yeastadhesins.Wehavenotattemptedtoaddressallareasof
S
these cases, deletion of some amino acids from this region the adhesin literature. Notably absent, for example, is a dis- T
A
results in inactive adhesins (26, 52, 85, 106). In an elegant cussionofthelargeandgrowingbodyofliteratureonthemany T
study,FriemanandCormackshowedthatwhenthisregionwas regulatory pathways that govern expression of the adhesins E

deleted from Eap1p, the adhesion domain was present in the (for reviews, see references 71, 74, 76, 91, and 124). FLO11 U
N
wall but was not sufficiently exposed to bind to its ligand be- gene expression, for example, is regulated by many signaling I
V
causethestalkwastooshort(26). pathways (94), including the cyclic AMP/protein kinase A
Two features of the Ser/Thr-rich C-terminal regions are pathway (2, 70, 95–97, 102, 127), a mitogen-activated protein
commonbuthaveunknownconsequences,andtheirroleshave kinasepathway(29,86,104,121),nutritionalsensingpathways
not been systematically tested. First, all Ser/Thr-rich regions (8, 17, 37, 68, 84, 120), a quorum-sensing pathway (12), and
alsohavesubstantialclustersofhydrophobicresidues(Fig.1). cyclins (83). Perhaps because of this, the FLO11 promoter is
Second, the C-terminal regions of the Ser/Thr regions are one of the largest known in yeast, as befits a gene that must
often rich in Cys residues as well. These Cys residues form a respondtodiversecircumstances.
disulfidenetworknearthesurfaceofthewall,andthisnetwork The common structural motifs of the adhesins described
limits wall permeability (Fig. 1) (20, 63, 64). Such a region in here suit adhesin function extremely well. The glycoproteins
Aga1phelpstocross-linktheadhesintothewall,presumably are secreted to the outer face of the plasma membrane, then
throughbondstoCysresiduesinotherGPI-anchoredproteins they are covalently anchored in the wall with their binding
(52,64,106). domains elevated beyond the wall surface (10, 26, 79). Their